Polymorphism of BF,C2, and GLO in Japanese patients with insulin-dependent diabetes mellitus: Confirmation of an increase of BF *FT

Summary The distribution of phenotypes controlled by three HLA-linked loci BF, C2, and GLO has been studied in Japanese patients with insulin-dependent diabetes mellitus (IDDM). A slight but significant higher incidence of a rare varian BF ^*FT (=^* F075) in patients was confirmed in the combined data with our previous study (Tokunaga et al. 1981 b). No significant association of C2 and GLO alleles with IDDM was found.     

The susceptibility to insulin-dependent diabetes mellitus bis associated with C4 allotypes independently of the association with HLA-DQ alleles in HLA-DR3, 4 heterozygotes

In the genetically homogeneous Danish population, 27 HLA-DR3,4 heterozygous patients with insulin-dependent diabetes mellitus (IDDM) and 19 DR3,4 heterozygous controls without family history of IDDM were investigated for HLA-region markers and Gm and Km immunoglobulin allotypes. The aim was to define susceptibility factors for IDDM development other than HLA-DR using a number of techniques: lymphocytotoxicity (HLA-DR and DQ antigens), cellular methods (Dw and DP typing), restriction fragment length polymorphism (DQ alleles), electrophoresis and immunofixation (BF and C4 allotypes), and passive hemagglutination inhibition (Gm and Km immunoglobulin allotypes). The complement allotype C4A3 and the HLA-DQw8 (DQw3.2) antigen were found in all of the patients, whereas this was the case for only 8 of the 19 controls (P=6 x 10^–6): five lacked C4A3, five others lacked DQw8, and one of the controls lacked both of these factors. Fourteen of the patients had the complement allotype C4B3 versus three of the controls (P=0.01). Previously reported family studies suggest that these alleles are part of the following haplotype: B15, BFS, C4A3, C4B3, DR4, Dw4, DQw8, and these factors were found together in ten of the patients versus one of the controls (P=0.01). The markers usually associated with DR3 did not show significant differences between IDDM patients and controls, and the non-HLA markers studied showed no significant deviation from what was expected. In addition to the susceptibility factor DQw8, the study suggests the existence of susceptibility genes for IDDM near the complement C4 genes on DR4-carrying haplotypes. Since recent works have shown that the structural gene for the monokine tumor necrosis factor alpha (TNF-) is located between the HLA-B and C4 loci and that TNF- might be of importance in IDDM pathogenesis, the hypothesis is put forward that the C4-associated IDDM susceptibility reflects linkage dis-equilibrium between the C4 gene and a gene controlling TNF- production. The high relative risk for IDDM in HLA-DR3,4 heterozygotes might be explained by the combined action of IDDM-specific susceptibility genes on DR4 haplotypes and DR3-linked susceptibility genes associated with predisposition to autoimmunity.     

Apparently non-expressed alleles of factor B (BF) code for hypomorphic proteins

In three families with an apparent non-expressed factor B (BF) allele (BF ^* Q0), advanced methods of isoelectric focusing for the determination of BF F subtypes revealed different hypomorphic BF products (BF QL) with functional hemolytic activity expressed by the assumed BF ^* Q0 allele. A Taq I and a Msp I restriction fragment length polymorphism as well as the Ba fragment of the expression products showed banding patters for the BF ^* QL alleles corresponding to BF S types, whereas an altered Bb fragment was seen in two BF QL products. In one family an intragenic recombination site within the Bb part of the BF gene was assumed. Investigations of factor B and its conversion fragments, as demonstrated by the used methods, allow to complement molecular genetic investigations of BF ^* Q0 alleles in heterozygous genotypes on a protein level. We conclude that apparently non-expressed alleles of factor B code for hypomorphic but functionally active proteins. Correspondence to : I. Siemens.     

Human BF*F-subtypes: segregation analysis with inclusion of MHC haplotypes

Summary The segregation of factor B(BF)F subtypes was analyzed in conjunction with other MHC markers in 15 families with 89 offspring. Informative data for BF F subtypes were obtained from 11 families, 6 of them with known recombinant individuals for the HLA-B/DR/GLO region. The subtypes did not contribute further to the localization of the cross-overs, but followed the known segregation of conventional BF allotypes. In 2 families of one kinship, the recognition of heterozygous BF^*FAFB individuals could be established following the inclusion of three generations. The rarer of the two BF F subtype alleles, BF^*FA, is positively associated with the HLA haplotypes BW62, CW3, C4A^*3 and A29, CWX, B44, C4A^*3, B^*1, DR7. BF F subtypes are regarded as a very useful additional tool for studies of MHC organization and disease association.     

MHC-linked class III genes. Analysis of C4 gene frequencies,complotypes and associations with distinct HLA haplotypes in German Caucasians

The class III complement components, C4, C2 and factor B (BF), are encoded in the human major histocompatibility complex (MHC). The two genes determining C4 (C4A and C4B) display considerable polymorphism and, thus, are important markers for HLA. In combination with alleles of C2 and BF they can be grouped into unique complotypes. We have analyzed the C4 alleles in a panel of 204 unrelated German Caucasians and studied their segregation with HLA haplotypes in 24 normal families. Inclusion of the class III markers with the class I and 11 alleles provides a more refined picture of the genetic structure of the MHC in these families. When charted according to the HLA-B locus specificities the MHCs can be clustered into groups showing distinctly homogenous or heterogenous complotypes. The identification of such groups is valuable for the selection of genetic material to analyze the molecular genetics of the human MHC.Abbreviations BF factor B - C2 second component of complement - C4 fourth component of complement - EDTA ethylenediamine tetraacetate - GLO glyoxalase-I - MHC major histocompatibility complex     

Another family with a silent allele of properdin factor B polymorphism (BF*QO)

Summary In five of eight members of a three generation family the existence of a silent allele of the properdin factor B polymorphism (BF^*QO) was indicated by immunofixation of BF electrophoretic variants and by the hemolytic overlay after isoelectric focusing of BF allotypes. This was further supported by the results of HLA-A, B, C, DR, C2, C4A, C4B, GLO-typing. BF protein was decreased in all heterozygous BF deficient family members. The absolute hemolytic activity, however, was obviously compensated for by an increased relative functional activity of the normal S or F alleles on the other chromosome.     

Susceptibility to IDDM Is marked by MHC supratypes rather than individual alleles

107 patients with insulin-dependent diabetes mellitus (IDDM) were typed for HLA A, B, C, and DR antigens, and for complement C4A, C4B, and Bf alleles, and the results were compared with those of a combined reference group of 332 appropriately matched healthy subjects. Supratypes (allelic combinations) were identified from the phenotype of each group, and it was shown that the frequency of several supratypes is increased in patients with IDDM, in particular supratypes (A1 Cw7) B8 C4AQ0 C4B1 BfS DR3 (P = 0.0001), (A30 Cw-) B18 C4A3 C4BQ0 BfF1 DR3 (P = 0.0003), (A2 Cw3) B62 C4AR C4B2.9 BfS DR4 (P = 0.0002), and three other supratypes including DR4. It was also shown that increases in the frequency of individual alleles are secondary to increases in supratype frequency. Moreover, supratypes appeared to interact; the presence of two relevant supratypes being particularly important. The absolute risk of IDDM was approximately 0.5 in subjects who were homozygous for B18 C4A3 C4BQ0 BfF1 DR3. We concluded that genetic susceptibility is best recognized by MHC supratypes rather than isolated alleles, and that supratype combinations make the identification of even greater disease risk possible.     

Further study on a BF silent allele

Summary A family was found which indicated the existence of a silent allele (BF ^* QO) at the locus for complement factor B. Three generations with eight members were studied. Four individuals were considered to be heterozygous for B deficiency because of unusual segregation patters of the BF electrophoretic variants and low levels of B. Haplotype study on the other HLA-linked markers supported the presumption. No unusual products were detected by immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

There are two C4 genetic loci and a null allele in the chimpanzee

Genetic polymorphism in C4 in the chimpanzee was studied by agarose gel electrophoresis of desialated plasma and development of patterns by immunofixation with antiserum to human C4 and by a C4-sensitive hemolytic overlay. In general, immunofixation patterns showed multiple partially overlapping bands of which only the most cathodal had strong hemolytic activity. In analogy to human C4, the latter were designated C4B, whereas those detected by immunofixation which had little hemolytic activity were designated C4A. Chimp C4A and C4B reacted with human and mouse (monoclonal) anti-C4B and human anti-Ch1 but neither reacted with monoclonal anti-C4A or human anti-Ch2, Ch3, Rg1, or Rg2. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the alpha chain of C4B showed a slightly lower apparent relative mass than that of C4A at around M _r 93 000. There were three C4A variants and two C4B variants inherited in families as autosomal codominant traits, as C4A-C4B cosegregating pairs with no detectable crossing-over. These pairs were inherited with chimpanzee leukocyte antigen types C2 and BF variants without detectable crossing-over. Half-null C4 haplotypes with C4B ^*Q0 were observed in family studies. Nine BF, C2, C4A, C4B allelic haplotypic combinations (complotypes) were identified among presumably unrelated chimpanzees.Abbreviations used in this paper: ChLA chimpanzee leukocyte antigen - HLA human leukocyte antigen - EDTA ethylenediaminetetraacetate     

A combined segregation and linkage analysis of insulin-dependent diabetes mellitus.

In an effort to clarify the mode of inheritance of insulin-dependent diabetes mellitus (IDDM), a total of 230 nuclear families with pointers were analyzed using the computer program COMBIN. Each family was ascertained without deliberate selection for multiplex families, and most families were completely typed for HLA-B, HLA-DR, and properdin factor B (Bf). There were 186 families with normal parents, 44 families with one affected parent, and no families with two affected parents. The computer program COMBIN evaluates evidence for a major locus of disease susceptibility, linkage of the major locus to a known genetic marker locus, linkage disequilibrium between the marker haplotypes and disease susceptibility, pleiotropic effects, and presence of an unlinked modifier. The parameters of COMBIN are T, Q, and D, representing the displacement, gene frequency of the IDDM allele, and dominance, respectively, of the major locus--and TM, QM, and DM being the analogous parameters of the modifier. In addition, the recombination fraction, theta, between the IDDM locus and HLA as well as the coupling frequencies are estimated. Finally, COMBIN simultaneously performs segregation and linkage analysis, with the optimal model being adjusted by the fit to the haplotype sharing distribution of IDDM. The results of these analyses indicated that the best-fitting genetic model of diabetic susceptibility appears to be a single major locus with near recessivity on a scale of standardized genetic liability, with gene frequency of the IDDM susceptibility allele of approximately 14%. In addition, the recombination fraction between the major locus and HLA is zero in all models; that is, for the B-BF-DR haplotype, the IDDM locus is tightly linked, probably (according to data from previous studies) to HLA-DR. Information determined by magnitude of coupling frequencies indicated that there is significant positive linkage disequilibrium with the haplotypes B8-BfS-DR4 and B15-BfS-DR4, significant negative linkage disequilibrium with B7-BfS-DR2, and intermediate disequilibrium for B8-BfS-DR3, B18-BfF1-DR3, and B40-BfS-DR4. Significant evidence in favor of an unlinked (to HLA) modifier (either single major locus or polygenes) could not be demonstrated. In conclusion, genetic susceptibility to IDDM appears to be most consistent with a single major locus with near recessivity that is tightly linked to HLA.     

Molecular characterization of MHC class II antigens (β 1 domain) in the BB diabetes-prone and -resistant rat

The BB or BB/Worcester (BB/W) rat is widely recognized as a model for human insulin-dependent diabetes mellitus (IDDM). Of at least three genes implicated in genetic susceptibility to IDDM in this strain, one is clearly linked to the major histocompatibility complex (MHC). In an attempt to define the diabetogenic gene(s) linked to the MHC of the BB rat, cDNA clones encoding the class II MHC gene products of the BB diabetes-prone and diabetes-resistant sublines have been isolated and sequenced. For comparison, the ₁ domain of class II genes of the Lewis rat (RTl^L) were sequenced. Analysis of the sequence data reveals that the first domain of RT1.D and RT1.B chain of the BB rat are different from other rat or mouse class 11 sequences. However, these sequences were identical in both the BB diabetes-prone and BB diabetes-resistant sublines. The significance of these findings is discussed in relation to MHC class II sequence data in IDDM patients and in the nonobese diabetic (NOD) mouse strain.     

Linkage disequilibrium of HLA-SB1 with the HLA-A1, B8, DR3, SCO1 and of HLA-SB4 with the HLA-A26, Bw38, Dw10, DR4, SC21 extended haplotypes

Homozygous typing cells from 13 normal HLA-A1, B8, Dw3, DR3 and five normal HLA-A26, Bw38, Dw10, DR4 individuals were typed for the following markers: HLA-SB, MB, MT; complement proteins BF, C2, C4A, C4B; and GLO. Ninety-one percent of A1, B8, Dw3, DR3 homozygous individuals (HI) tested were homozygous for BF ^* S, C2 ^* C, C4A ^* QO, and C4B ^*1 (SCO1 complotype), which indicates that the SCO1 complotype is in linkage disequilibrium with the A1, B8, DR3 haplotype in randomly selected normal populations. Sixty-seven percent of HLA-A1, B8, Dw3, DR3, SCO1 positive HI also expressed SB1; since the frequency of SB 1 in random Caucasian populations is 11.2%, this finding indicates that SB1 is in linkage disequilibrium with the A1, B8, DR3, SCO1 extended haplotype. All HI with the A26, Bw38, Dw10, DR4 haplotype were homozygous for both SC21 and SB4, suggesting that SC21 and SB4 should be included in the A26, Bw38, Dw10, DR4 extended haplotype. On the other hand, neither of the GLO markers were found in association with either haplotype. The results of this study indicate that HLA-SB is included in some extended haplotypes and may be important in these markers for diseases such as insulin-dependent diabetes mellitus. This study also demonstrated an apparent influence of HLA-SB on primary mixed lymphocyte culture (MLC) responses. The mean relative response of primary MLCs between individuals matched for HLA-A, B, D, DR, MB and MT but not SB was 40% of that for the MLCs with mismatched HLA-D, significantly higher than the MLCs matched for all HLA and complotypes.     

The location ofC2, C4, andBF relative toHLA-B andHLA-D

The loci forHLA-A, B, C, D, andDR are known to be closely linked to the structural loci for the complement components C2, BF, and the duplicated loci for C4, C4A and C4B. Conflicting evidence has been presented for the order of these genes. However, new techniques have made possible identification of markers in theHLA-D andC4 region for nearly all identified haplotypes. In our population we have confirmed fiveHLA-B-D crossovers and in each case informative allotypes of C2, BF, or C4A and C4B segregated withHLA-D orDR suggesting that the loci for these proteins lie close toHLA-D andDR. These findings may be of importance for resolving problems encountered in the assignment ofHLA-D alleles.     

Heterogeneity of HLA genetic factors in IDDM susceptibility

The association of certain HLA-D alleles with insulin-dependent diabetes mellitus (IDDM) is well known. One hundred and sixty-one non-related diabetic individuals and 142 non-related healthy controls were typed for the HLA DR-DQw-Dw association, using a restriction fragment length polymorphism (RFLP) typing method that combines three probe/enzyme systems: DRB/Taq I, DQB/Taq I, and DQB/Bam HI. Comparison of frequencies in both diabetics and controls confirms previous results in terms of HLA class II and IDDM association. Moreover, we have found that DR3/4 heterozygous individuals are more susceptible to IDDM when they are also Dw25 (associated with B18) than when they are Dw24 (associated with B8). Using oligonucleotide dot-blot hybridizations we analyzed the HLA-DQB1 sequence of DR3, Dw24 and DR3, Dw25 homozygous individuals, and we found no difference at position 57 between these two DR3-carrying haplotypes. This observation points to the heterogeneity of HLA genetic factors in IDDM susceptibility. Offprint requests to: D. Cohen.     

Genetic markers for glutamic acid decarboxylase do not predict insulin-dependent diabetes mellitus in pairs of affected siblings

Reliable genetic and immunological markers are important in the prediction of insulin-dependent diabetes mellitus (IDDM). Since glutamic acid decarboxylase (GAD) is a candidate primary autoantigen, we examined the possible linkage between IDDM and the genes encoding GAD₆₅ (GAD2, 10p11–12) and GAD₆₇ (GAD1, 2q31) in 58 Danish IDDM affected sib pairs. The allelic inheritance of 10 polymorphic dinucleotide repeat sequences spanning the chromosomal regions of the two GAD genes, were examined by affected sib pair analysis (ASP). In addition a restriction fragment length polymorphism (RFLP) was identified in the gene encoding GAD₆₅ using the restriction enzyme PvuII. The GAD gene markers were analyzed in relation to the presence of specific HLA types and GAD autoantibodies. No evidence of linkage was found between IDDM and either of the genes encoding GAD. This was also the case when subgroups carrying specific HLA susceptibility alleles were analyzed. Nor did we observe any association between these GAD genetic markers and the presence of GAD autoantibodies. Considering the high prevalence of GAD autoantibodies in IDDM, a putative genetic association between GAD and IDDM would be expected to affect most diabetic individuals. Therefore, our data indicate that the association between GAD and IDDM is not genetically determined, and that microsatellites used in this study do not contribute to the prediction of IDDM. Received: 1 July 1996 / Revised: 21 August 1996     

Partial C4 deficiency in subacute sclerosing panencephalitis

In an immunogenetic study, 23 subacute sclerosing panencephalitis (SSPE) patients and their families were studied for the HLA region markers HLA-A, B, C, DR, BF, C2, C4A, C4B, GLO I, and PGM3. In addition, C3, C4, and factor B serum levels were determined. A highly significant association of C4A^*QO with SSPE was found. Furthermore, two rare haplotypes, C4A*QOB*9QO, two C4ACh+ allotypes, and four Ch partial inhibitors were detected, which possibly impair the function of the C4 molecules. HLA-DR5 was increased. In addition, a number of rare HLA-A, C, B, DR haplotypes were observed. It is postulated that rare C4 molecular deficiency might be a predisposing factor in the pathogenesis of SSPE.     

Murine complement factor B (BF) Sexual dimorphism and H-2-Linked polymorphism

Polymorphism of murine BF is described using agarose gel electrophoresis of EDTA-plasma. The proteins were blotted onto cellulose nitrate sheets and BF was detected by incubation of these sheets with anti-BF serum, anti-IgG serum, and ¹²⁵I-labeled protein A successively. After autoradiography, four or five main BF bands were found in plasma of male mice. The strain WLL/BrA (H-2 ^bs carried a more anodal variant than the strains 020/A (H-2 ^pz , B10 (H-2 ^b , B10.A (H-2 ^a , B10.M (H-2 ^f ), and OIR (H-2 ^q ). In backcross and F₂ generations the BF variants always cosegregated with the H-2 haplotypes. In this way linkage to H-2 could be established. When the electrophoretic BF patterns of males and females were compared, a sexual dimorphism was discovered; the females of each strain had only three main BF bands compared with the four or five found in males. However, no differences in level between males and females could be detected, probably because the three BF bands in the females were stronger. These data extend the information on the interspecies homology of the MHC and may open new possibilities for studies of the genetic organization and hormonal regulation of the H-2 complex.     

Cloning and sequencing of the porcine complement factor B

A genomic clone, SSBf1, containing the complement factor B (BF), a major histocompatibility class III antigen, has been isolated from a porcine genomic library. Partial sequencing and comparison with a human BF gene has identified seven exons coding for amino acids of Ba and Bb, the two subunits of BF. The protein sequence similarity with the human BF is on the average 87%. Southern blot analysis confirmed the existence of only one BF gene per haploid genome. Restriction fragment length polymorphism typing with Taq I showed that there are at least three different porcine BF-haplotypes. Address correspondence and offprint requests to: Luc J. Peelman.     

High-resolution linkage mapping for susceptibility genes in human polygenic disease: Insulin-dependent diabetes mellitus and chromosome 11q

Insulin-dependent diabetes mellitus (IDDM) has a complex pattern of genetic inheritance. In addition to genes mapping to the major histocompatibility complex (MHC), several lines of evidence point to the existence of other genetic susceptibility factors. Recent studies of the nonobese diabetic mouse (NOD) model of IDDM have suggested the presence, on mouse chromosome 9, of a susceptibility gene linked to the locus encoding the T-cell antigen, Thy-1. A region on human chromosome 11q is syntenic to this region on mouse chromosome 9. We have used a set of polymorphic DNA markers from chromosome 11q to investigate this region for linkage to a susceptibility gene in 81 multiplex diabetic pedigrees. The data were investigated by maximization of lod scores over genetic models and by multiple-locus affected-sib-pair analysis. We were able to exclude the presence of a susceptibility gene (location scores less than -2) throughout greater than 90% of the chromosome 11q homology region, under the assumption that the susceptibility factor would cause greater than 50% of affected sib pairs to share two alleles identical by descent. Theoretical estimates of the power to map susceptibility genes with a high-resolution map of linked markers in a candidate region were made, using HLA as a model locus. This result illustrates the feasibility that IDDM linkage studies using mapped sets of polymorphic DNA markers have, both for other areas of the genome in IDDM and for other polygenic diseases. The analytic approaches introduced here will be useful for affected-sib-pair studies of other complex phenotypes.     

Heterogeneity of human C4 gene size

In this article we present a study showing that the human C4 genes differ in length because of the presence or absence of a 6.5 kb intron near the 5 end of the gene. DNA from individuals of known HLA, factor B, and C4 haplotypes was analyzed for restriction fragment length polymorphism (RFLP) by Southern blot analysis with C4-specific cDNA probes. The RFLP patterns obtained showed that the C4 genes are either 22.5 kb or 16 kb in length. They are referred to as long and short C4 genes, respectively. A population study was carried out to examine the distribution of the gene size according to C4 allotypes and haplotypes. Long C4 genes included all C4A genes studied and also some C4B allotypes, e. g., B1 on most C4 A3B1 haplotypes. Similarly, C4B null genes were found to be of the long form. Other C4B allotypes tested were found to be coded for by short C4 genes, including B2, B1 in C4 A6B1 and C4 AQOB1 (with a single C4B gene haplotype).Abbreviations used in this paper C4 fourth component of complement - C2 second component of complement - BF factor B - MHC major histocompatibility complex - RFLP restriction fragment length polymorphism - EDTA ethylenediaminetetraacetic acid - SDS lauryl sulfate, sodium salt